The invention relates to methods for identifying inhibitors of fatty acid biosynthesis in bacteria.
Fatty acid biosynthesis (FAB) is necessary for the production of bacterial cell walls, and therefore is essential for the survival of bacteria (Magnuson et al., 1993, Microbiol. Rev. 57:522-542). The fatty acid synthase system in E. coli is the archetypal type II fatty acid synthase system. Multiple enzymes are involved in fatty acid biosynthesis, and genes encoding the enzymes fabH, fabD, fabG, acpP, and fabF are clustered together on the E. coli chromosome. Clusters of FAB genes have also been found in Bacillus subtilis, Haemophilus influenza Rd, Vibrio harveyi, and Rhodobacier capsulatus. Examples of FAB genes in B. subtilis include fabD, yjaX and yhjB (encoding synthase III), fabG, ywpB, yjbW, yjaY, ylpC, fabG, and acpA. The ylpC, fabG, and acpA genes are contained within a single operon that is controlled by the PylpC promoter.
Using genetic and biochemical methods, the targets and mechanisms of action of several inhibitors of the elongation part of the FAB pathway have been identified (FIG. 1). The FAB inhibitor cerulenin inhibits xcex2-ketoacyl-ACP synthase, and the inhibitor thiolactomycin inhibits acetoacyl ACP synthase (Omura, 1981, Meth. Enzymol. 72:520-532; Moche et al., 1999, J. Biol. Chem. 274:6031-6034; and Jackowski et al., 1989, J. Biol. Chem. 264:7624-7629). The FAB inhibitors isoniazid, triclosan, and diazaborine inhibit enoyl-acyl carrier protein reductase (Quemard et al., 1995, Biochem. 34:8235-8241; McMurray et al., 1998, Nature 394:531-532; Heath et al., 1998, J. Biol. Chem. 273:30316-30320; Heath et al., 1999, J. Biol. Chem. 274:11110-11114; Levy et al., 1999, Nature 398:383-384; and DeBoer et al., 1999, Mol. Microbiol. 31:443-450). In Pseudomonas aeruginosa, triclosan has been shown to inhibit the enoyl-acyl carrier protein reductase (FabI), which converts trans-2-enoyl-ACP to acyl-ACP (Hoang and Schweizer, supra). The FAB pathway provides the acyl groups for production of acylated homoserine lactones (HSLs). HSLs are the signaling molecules involved in quorum sensing, i.e., bacterial cell-to-cell signaling, in a wide variety of bacteria. In pathogenic bacteria, such as Pseudomonas, quorum sensing is a mechanism for regulating the expression of virulence factors (Hastings and Greenberg, 1999, J. Bacteriol. 181:2667-2668).
The invention is based upon the discovery that the activity of promoters of certain genes is increased in the presence of compounds that inhibit B. subtilis FAB. Thus, compounds that inhibit FAB can be identified by their ability to increase the activity of the B. subtilis PyhfB and PylpC promoters. Various promoters can be used in the invention, provided that the activity of the promoter is upregulated by a FAB inhibitor, such as cerulenin or triclosan. FAB inhibitors that slow the growth of, or kill, bacteria are candidate antibacterial agents that can be used in methods of treating bacterial infections. The invention thus provides a rapid and convenient method for identifying (i) compounds that inhibit FAB and which can subsequently be derivatized to produce antibacterial agents, as well as (ii) compounds that inhibit FAB and which are antibacterial agents. Because the FAB pathway is involved in the synthesis of HSLs, the invention also provides a method for identifying compounds that inhibit HSL synthesis. Such compounds can be used to inhibit bacterial virulence. If desired, such inhibitors of HSL synthesis can be further derivatized using standard medicinal chemistry techniques to produce inhibitors of virulence having increased potency.
Accordingly, the invention features a method for determining whether a test compound is an inhibitor of bacterial FAB. The method includes: (i) contacting a bacterial cell with a test compound, wherein the bacterial cell contains (a) a promoter (e.g., PyhfB or PylpC), the activity of which is increased in the presence of a compound that inhibits FAB, operably linked to (b) a reporter gene; and (ii) measuring activity of the promoter, wherein an increase in activity, relative to the level of activity of the promoter in the absence of the test compound, indicates that the test compound is an inhibitor of bacterial FAB.
The invention also includes a method for determining whether a test compound is an antibacterial agent, the method comprising: (i) contacting a bacterial cell with a test compound, wherein the bacterial cell contains (a) a promoter (e.g., PyhfB or PylpC), the activity of which is increased in the presence of a compound that inhibits FAB, operably linked to (b) a reporter gene; (ii) measuring activity of the promoter, wherein an increase in activity, relative to the level of activity of the promoter in the absence of the test compound, indicates that the test compound is an inhibitor of FAB; and (iii) determining whether the compound is an antibacterial agent by determining whether the compound kills, or slows the growth of, bacteria. Optionally, the test compound may be further assayed in a biochemical assay (e.g., in an extract of the cell) to determine which step in the pathway is inhibited, and to confirm that the test compound inhibits fatty acid biosynthesis. For example, inhibition of fatty acid biosynthesis can be detected as inhibition of incorporation of acetate into fatty acids or phospholipids. Conventional methods can be used to measure inhibition of incorporation of acetate.
An increase in activity of the promoter can be measured, for example, by measuring expression of a reporter gene that is operably linked to the promoter, such as a lacZ cat, gus, a green fluorescent protein gene, or a luciferase gene. Other suitable reporter genes are well known in the art and can be used in the invention. If desired, the activity of the promoter can be measured by measuring binding of antibodies to a product of the reporter gene (e.g., a protein encoded by the reporter gene), with an increase in the level of bound antibodies reflecting an increase in activity of the promoter. Alternatively, activity can be measured by measuring the level of mRNA transcribed from the reporter gene, with an increase in the mRNA level reflecting an increase in promoter activity.
The invention also provides methods of preparing (i) an inhibitor of fatty acid biosynthesis and/or (ii) an antibacterial agent. The methods include: screening multiple test compounds by the methods described above; identifying candidate compounds that upregulate promoter activity; isolating one or more lead compounds from the candidate compounds; identifying and selecting a lead compound that inhibits fatty acid biosynthesis or bacterial growth; and formulating the selected lead compound as an inhibitor of fatty acid biosynthesis or as an antibacterial agent. A xe2x80x9clead compoundxe2x80x9d is a test compound that increases promoter activity by at least 3 times the standard deviation, plus the mean. If desired, lead compounds can subsequently be derivatized using conventional medicinal chemistry methods, as described herein.
Similarly, the invention features methods for preparing (i) an inhibitor of FAB or (ii) an antibacterial agent. The methods include screening multiple test compounds by the methods described above; identifying candidate compounds that upregulate promoter activity; isolating one or more lead compounds from the candidate compounds; derivatizing the lead compound(s), thereby producing a derivative of the lead compound; identifying derivatives that inhibit FAB or bacterial growth; and formulating the derivative as an inhibitor of FAB or as an antibacterial agent (e.g., by admixture with a pharmaceutically acceptable carrier). Inhibitors of FAB and antibacterial agents prepared by such methods also are included within the invention. Such compounds can be used in methods for inhibiting bacterial fatty acid biosynthesis or growth of bacteria in an organism having a bacterial infection.
The invention also provides a method for identifying an inhibitor of bacterial evirulence. In such a method, a FAB inhibitor identified by the methods disclosed herein can be further tested for its ability to inhibit the synthesis of HSL. Inhibition of acylated homoserine lactone synthesis can be detected as inhibition of enoyl-acyl carrier protein reductase activity. In a convenient assay, the FAB inhibitor can be tested in vitro for its ability to inhibit the synthesis of N-butyryl-L-homoserine lactone (C4-HSL) in the presence of FabI (enoyl-acyl carrier protein reductase), the HSL synthetase RhII, the cofactor NADH, and the substrates crotonyl-ACP and S-adenosylmethionine (see, e.g., Hoang and Schweizer, supra). A decreased level of HSL synthesis in the presence of the test compound, relative to the level of HSL synthesis in the absence of the test compound, indicates that the FAB inhibitor is an inhibitor of HSL synthesis. Because HSLs regulate the expression of virulence factors, inhibitors of HSL synthesis can be used to inhibit bacterial virulence, e.g., in a method for treating a bacterial infection. If desired, such HSL synthesis inhibitors can be derivatized, using standard medicinal chemistry techniques as described herein, to produce derivatives that have increased potency as inhibitors of virulence in bacteria.
The invention offers several advantages. For example, various embodiments of the invention can readily be used for high-throughput screening (HTS) of a wide variety of test compounds. Thus, lead compounds can readily be selected from a large number of test compounds. Assays employing the PyhfB and PylpC promoters are capable of detecting FAB inhibitors at concentrations both above and below their minimal inhibitory concentration (MIC). Thus, the assays described herein provide a high level of sensitivity and are expected to detect growth inhibitory (i.e., bacteriocidal) compounds, as well as less potent inhibitors of the FAB pathway, which can be subsequently modified using standard medicinal chemistry techniques and by evaluating structure-activity relationship (SAR) data. Because the assays are cell-based, the assays identify antibacterial agents that can efficiently enter bacterial cells. Thus, the assays allow the identification of potent antibacterial compounds and compounds of structural interest that may have relatively modest potency, but have favorable cell permeability properties. In addition, because FAB is a biochemical pathway, the methods disclosed herein enable the identification of compounds that may inhibit any enzymatic function or step in the pathway.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, technical manuals, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.